β-Galactosidase has been a workhorse as a label for detection of a wide variety of ligands in numerous different contexts. There have been numerous applications of β-galactosidase as an intact enzyme and as fragments that when combined form an active enzyme. When fragments are used, the assay is referred to as “Enzyme Fragment Complementation” (“EFC”) assay. The use of fragments can be divided into two different situations: (1) the fragments have sufficient affinity for each other that they bind independently to form an active enzyme; and (2) the fragments have a sufficiently low affinity for each other that they require an enhancement for binding, such as each of the fragments being bound to entities that do bind to each other, where when the entities bind an active enzyme is obtained. Generally, arbitrarily one of the fragments will be called an enzyme donor fragment (“ED”) and the other fragment will be called an enzyme acceptor fragment (“EA”). In the case of (1) the smaller fragment is referred to as the ED.
In the first case, numerous assays have been developed where an agent is present that can inhibit the binding. In the second case, one requires that the entities bind independently or an agent is present that enhances the binding of the entities. Each of these situations has found extensive application as evidenced by the numerous patents that have issued covering the use of EFC.
Since one is dealing with an enzyme, large signal amplifications can be achieved. The signal amplification must be considered in light of the background. Even with the low affinity fragments, the relatively high concentration(s) of one or both of the fragments can result in a background signal. Depending on the nature of the assay, results can vary with different ED fragments, where the fragments may interfere with the protein to which a fragment is bound, differences may be observed between different proteins as to the degree of activity in the case of stimulation and absence of stimulation, the degree of background activity, and the like.
Small differences in structure may have significant differences as to the manner in which a fragment may be used as was observed in U.S. Patent application no. 2005/0287522. It is found that depending upon the nature of the ED, different structures can have significant effects on the utility of an assay. Therefore, it is not a matter of one structure fits all. Rather, it is found that for particular situations one ED will provide a robust assay while another will be inoperative. It has now been found that particular sequences find application in particular assays, rather than providing a universal fragment that will result in a commercially acceptable assay for all or most ligands.